biacore x100 gs bookwater vials, 4 ml (package of 1000 vials) br-1006-54. biacore x100 getting...

GE Healthcare

Biacore X100

Getting Started

2 Biacore X100 Getting Started 28-9615-81 Edition AA

Contents

Contents

Biacore X100 Getting StartedIntroduction .............................................................................................. 5

Requirements ...............................................................................................................5References .....................................................................................................................5Contents of the Getting Started Kit .....................................................................6Ordering information ................................................................................................7

Background informationBiacore terminology ................................................................................ 9Sensor surface properties .................................................................... 10Immobilization ........................................................................................ 11

Amine coupling.........................................................................................................11Conditions for immobilization............................................................................11Immobilization pH-scouting ...............................................................................12Immobilization levels .............................................................................................15Ligand requirements..............................................................................................15

Interaction analysis ............................................................................... 16Sample requirements ............................................................................................16

Regeneration .......................................................................................... 17Testing regeneration conditions .......................................................................18Choice of regeneration conditions ...................................................................20

Instrument start upStart the system ..................................................................................... 21Buffer preparation and loading ........................................................... 22Inserting, removing or changing the sensor chip ............................ 23Preparing and loading samples and reagents ................................. 24

Biacore X100 Binding AssayDescription of the experimental set up ............................................. 25

Experimental materials.........................................................................................25Experimental steps .................................................................................................25Requirements ............................................................................................................26References ..................................................................................................................26

Binding Assay set up ............................................................................. 27Instrument preparations ......................................................................................27Binding Assay workflow and immobilization set up................................27Binding Analysis set-up.........................................................................................32

Evaluation of Binding data ................................................................... 37Examine the sensorgrams...................................................................................37Examine the predefined plots ............................................................................39Examine the results as a Bar Chart .................................................................41End your evaluation ...............................................................................................42

Biacore X100 Single Cycle Kinetics AssayDescription of the experimental set up ............................................. 43

Biacore X100 Getting Started 28-9615-81 Edition AA 3

Contents

Experimental materials.........................................................................................43Experimetal steps....................................................................................................43Requirements ............................................................................................................43References ..................................................................................................................43

Kinetics Assay set up .............................................................................44Instrument preparations ......................................................................................44Kinetic Assay workflow and immobilization set up..................................44Kinetic Assay set-up ...............................................................................................49

Evaluation of Kinetic data ....................................................................54


Biacore X100 Getting Started


IntroductionThis Getting Started handbook is designed as a self-study guide to introduce you to the basic operation of BiacoreTM X100, Biacore X100 Control Software and Biacore X100 Evaluation Software.

The handbook, together with the associated reagent kit , will take you through the basic steps in setting up and evaluating a binding analysis and a kinetics analysis using the Single Cycle Kinetics approach. Step-by-step instructions for the binding analysis experiment are also provided in the Support Navigator in the Biacore X100 Software.

The reagents in the Getting Started Kit are for training purposes only and GE Healthcare Bio-sciences AB can accept no responsibility for results obtained with these reagents in any other context.

The reagents should be stored at between +4 and +8°C and should be used within one week of opening.

RequirementsThe following are required for completing the Getting Started exercises:

• Biacore X100 instrument with Reagent Rack

• Familiarity with PC and Windows

• Getting Started Kit

• Sensor Chip CM5 (not included in the Getting Started Kit)

• Amine Coupling Kit (not included in the Getting Started Kit)

• Micropipettes; 2-10 µl, 10-100 µl and 100-1000 µl and tips

ReferencesFor further details on the topics discussed in this booklet, refer to:

• Biacore X100 Handbook

• Biacore Advisor Tutorial (CD-ROM)

• Sensor Surface Handbook


Biacore X100 Getting StartedIntroduction

Contents of the Getting Started KitThe contents of the Getting Started Kit are listed in Table 1. All solutions, except for the 10X HBS-EP+ buffer, ligand and analyte are ready for use. The 10X HBS-EP+ buffer, ligand and analyte should be diluted as described in the exercises and used immediately after dilution.

Table 1. Contents of the Getting Started Kit. For in vitro use only. Storage: +4 to +8°C for all solutions.

Reagent/Item, quantity Specification

10X Running buffer (50 ml) 10X HBS-EP+ buffer; 0.1 M HEPES pH 7.4, 1.5 M NaCl, 30 mM EDTA, 0.5% (v/v) Surfactant P20

Ligand (50 µl) Monoclonal mouse-anti-human 2-microglobulin

Analyte (50 µl) Human 2-microglobulin

Immobilization buffer (1 ml), 2 pcs

10 mM sodium acetate, pH 5.0

Regeneration solution (1 ml), 2 pcs

10 mM glycine-HCl, pH 2.5

Plastic vials and ventilated caps, 25 pcs of each

1.5 ml polypropylene microvials and ventilated kraton G (SEBS) rubber caps

Water vials, 2 pcs 4 ml polypropylene microvials



Ordering informationOrdering information is given in Table 2. For further information, please visit www.biacore.com or contact your local GE Healthcare representative.

Table 2. Ordering information.

Item Code No.

10X HBS-EP+ buffer (4 x 50 ml) BR-1008-26

Sensor Chip CM5 (package of 3 chips)Sensor Chip CM5 (package of 1 chip)

BR-1000-14BR-1003-99

Amine Coupling Kit BR-1000-50

BIAmaintenance Kit BR-1006-66

Biacore Advisor Tutorial, CD-ROM BR-1005-44

Sensor Surface Handbook BR-1005-71

Plastic vials, 11 mm (package of 500 vials) BR-1002-87

Caps for 11 mm vials (package of 400 caps) BR-1004-11

Water vials, 4 ml (package of 1000 vials) BR-1006-54


Biacore X100 Getting StartedIntroduction


Background information


This chapter is intended to provide the basis for a more detailed understanding of the main steps in a Biacore assay. The information goes beyond what is explicitly covered in the exercises and is, therefore, a complement to the exercises.

Biacore terminology• When molecular interactions are studied in Biacore, one of the

interactants is immobilized on the sensor surface while the other is passed over the surface in solution. In Biacore terminology, ligand refers to the immobilized component and the interactant present in the sample injected over the surface is referred to as the analyte (Figure 1).

Figure 1. “Ligand” refers to the immobilized interactant and “analyte” refers to the other interactant present in the sample injected over the sensor surface.

• The response is measured in resonance units (RU) and is proportional to the molecular mass on the surface. For an interactant of a given mass, therefore, the response is proportional to the number of molecules at the surface.

• A sensorgram is a plot of response against time, showing the progress of the interaction. The sensorgram is displayed on the computer screen during the course of an analysis.


Background informationSensor surface properties

There are three major steps in a Biacore assay. These are:

1 Immobilization: The process by which the ligand is attached to the sensor chip surface.

2 Interaction analysis: The analyte is injected over the sensor chip surface and the interaction between the analyte and the immobilized ligand is monitored.

3 Regeneration: The process of removing bound analyte from the ligand on the surface.

Sensor surface propertiesSensor chip CM5, which is used in the Getting Started Kit, is a glass slide coated with a thin layer of gold, to which a matrix of carboxymethylated dextran is covalently attached (Figure 2). The gold is required for generation of the surface plasmon resonance (SPR) response. The dextran matrix allows covalent immobilization of biomolecules using well-characterized chemistry and provides a hydrophilic environment suitable for a wide variety of protein interactions.

Figure 2. Schematic illustration of the structure of the surface of Sensor Chip CM5.

In addition to Sensor Chip CM5, which is the most versatile chip, GE Healthcare offers a range of sensor chips with different properties that allow immobilization of biomolecules with different characteristics. Refer to www.biacore.com for further information.

Surface matrixLinker layer

Gold film

Glass support



ImmobilizationThere are different ways of immobilizing substances to the sensor surface. The choice of immobilization method depends on the properties of the substance. The immobilization approaches may be directed towards amine, carboxyl, thiol or hydroxyl groups on the ligand, or may use specific tags introduced into the ligand.

Amine couplingAmine coupling chemistry is the most widely applicable approach for covalently attaching biomolecules to the sensor chip surface and is suitable for the ligand included in the Getting Started Kit. With this method, the dextran matrix on the sensor chip surface is first activated with a mixture of 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to give reactive succinimide esters. Ligand is then passed over the surface and the esters react spontaneously with amino groups or other nucleophilic groups to link the ligand covalently to the dextran (Figure 3). After the injection of ligand, ethanolamine is passed over the sensor surface to deactivate remaining active esters.

Figure 3. Amine coupling of ligands to the sensor chip surface.

Conditions for immobilizationAt the concentrations of ligand commonly used for immobilization (20-200 µg/ml), the expected levels of immobilized ligand would be low in the absence of a mechanism that attracts the ligand molecules to the sensor surface. The main mechanism for this concentration process is electrostatic attraction of the ligand to the surface. This attraction is referred to as pre-concentration and can result in a several thousand-fold concentration of ligand on the surface.

The carboxymethylated dextran matrix of the sensor chip carries a net negative charge at pH values above about 3.5. The pH of the immobilization buffer should, therefore, be higher than 3.5 and lower than the isoelectric point of the ligand in order to achieve efficient pre-concentration. For many proteins, coupling in 10 mM sodium acetate buffer (pH 4.5) works well, although the choice of pH can be a critical parameter in determining the success of


Background informationImmobilization

immobilization in some cases. If you have to use other conditions, bear the following considerations in mind:

• The ionic strength should be low (10 mM monovalent cations recommended) for the electrostatic attraction to occur.

• Buffer components containing primary amine groups and other strong nucleophilic groups (e.g. Tris or sodium azide) must be avoided for amine coupling, as these will compete with the ligand for active esters on the sensor chip surface.

Many proteins show limited stability in low ionic strength solutions and at low pH. The ligand solutions should, therefore, be prepared directly before use.

Immobilization pH-scoutingThe immobilization pH scouting procedure helps you to find the optimum pH for immobilization of proteins and some peptides. The pH can be important because efficient immobilization in many cases relies on electrostatic pre-concentration of the molecule on the surface. To perform pH scouting in Biacore X100, choose Other Options in the Biacore X100 Control Software, click Wizards and choose Immobilization pH Scouting. Follow the guidelines in the Support Navigator on how to proceed with the setup.

Example

In the example in Figure 4, the ligand analysed for suitable immobilization pH was the monoclonal anti-2-microglobulin antibody included in the Getting Started Kit. The ligand was diluted in sodium acetate buffers with different pH (pH 5.5, pH 5.0, pH 4.5 and pH 4.0) to a final concentration of 30 µg/ml in each sample.

After each injection, a wash solution was injected to remove any remaining ligand molecules. A short pulse of 1 M ethanolamine-HCl pH 8.5 (included in the Amine Coupling Kit) or 50 mM NaOH is commonly used for washing surfaces that have been used for pre-concentration/pH-scouting experiments.



As shown in Figure 4, the attraction of the protein into the dextran matrix on the sensor chip surface increased slightly, as indicated by the slope of the sensorgram, with decreasing pH of the buffers. This is explained by the fact that the protein is less positively charged at pH 5.5 as compared to pH 4.0, which affects the attraction to the negatively charged dextran matrix. pH 5.5, 5.0 and 4.5 all gave a satisfactory pre-concentration effect. At pH 4.0, the sensorgram approached a plateau at the end of the injection, which indicates that the maximum level of attraction was reached at that pH. In general, choose the highest immobilization pH that gives satisfactory preconcentration, to avoid exposing the ligand to unnecessarily low pH.

Note: It should be pointed out that pH-scouting does not provide a conclusive answer regarding suitable immobilization conditions. The chemistry of amine coupling is less efficient at low pH and in order to fully optimize the conditions and verify the choice of pH, the entire immobilization procedure should be performed.

Figure 4. Sensorgram showing the SPR responses generated by the ligand anti-2-microglobulin injected in immobilization buffer pH 5.5, pH 5.0, pH 4.5, pH 4.0, respectively.


Background informationImmobilization

In Figure 5, the sensorgrams from amine coupling of monoclonal anti-2-microglobulin at pH 5.5, 5.0, 4.5 and 4.0 are shown. Observe that the final immobilization level at pH 4 was lower than at the other pH values tested (Figure 4). This clearly demonstrates the pH-dependence of the amine coupling chemistry and pH 4.0 was excluded in this case.

As mentioned previously, the pre-concentration effects at pH 5.5, 5.0 and 4.5 were all acceptable (Figure 4). Since the immobilization level at pH 5.5 was higher than the immobilization levels at pH 5.0 and 4.5, pH 5.5 could appear to be the best choice (Figure 5). By looking at the sensorgrams (Figure 5), however, it becomes evident that at pH 5.5 the response after attraction and covalent coupling has not reached the maximum level. At pH 5.0, the sensorgram begins to flatten out after the covalent coupling (the maximum level is almost reached), which may contribute to an increase in the robustness of the assay set-up. At pH 4.5, the plateau is more evident, but at the same time the immobilization level has dropped. Consequently, in this case pH 5.0 should be chosen for immobilization of monoclonal anti-2-microglobulin.

Figure 5. Sensorgrams showing the immobilization of anti-2-microglobulin via amine coupling at pH 5.5, pH 5.0, pH 4.5 and pH 4.0, respectively.



Immobilization levelsThe binding capacity of the surface will depend on the levels of immobilized ligand. The term maximum response (Rmax) is often used in connection with Biacore experiments and describes the binding capacity of the surface in terms of the response at saturation. A theoretical Rmax value can be calculated using the formula below:

A theoretically calculated Rmax is often higher than the experimentally derived Rmax for the same interaction. There are many potential explanations for this, such as that the ligand is not fully active or that there is steric hindrance of the interaction.

Different applications may require different binding capacities. A low Rmax, for example, is often recommended in kinetic analyses, while higher levels are advantageous in concentration measurements.

Ligand requirementsThe quality and purity of the ligand have an important effect on determining the specificity and analyte binding capacity of the surface. The ligand should be as specific as possible for the analyte as the selectivity of the assay is determined by specificity. If necessary, an enhancement molecule can be used to increase the sensitivity and/or specificity of the assay.

The purity of the ligand is of vital importance for the experimental results. Impurities may be immobilized on the sensor chip and could affect the analyte binding capacity of the surface. Sensor surfaces immobilized with impure ligand solutions may give rise to results that are difficult to interpret. The recommendation is, therefore, to use as pure ligand solutions as possible.

Cross-reactivity (i.e. binding of analyte-related molecules to the ligand) and non-specific binding (i.e. general binding to the ligand) are other factors, which can be controlled through the choice of ligand. Note that these factors are generally applicable to the majority of interaction-based assay formats and are not specific to Biacore assays.

Rmaxanalyte MWligand MW------------------------------ immobilized amount stoichiometric ratio=


Background informationInteraction analysis

Interaction analysisDuring injection of the analyte, the binding of analyte to ligand takes place and is monitored in real time. After the analyte injection, when buffer flows over the surface, the dissociation of the analyte/ligand complex is monitored. Although analytes in crude sample matrices can be measured and the need for sample purification is low, a few guidelines on sample requirements are given below.

Sample requirementsThe detection principle in Biacore technology measures changes in refractive index (RI) that are related to changes in mass close to the sensor surface. The design of the opto-interface allows for measurements of samples in crude environments, such as serum and cell culture supernatants.

Sample environments that differ greatly from the running buffer will give rise to a bulk effect that is commonly present during an injection. Bulk refractive index effects do not affect the binding. Our recommendation is, however, that the samples should be diluted in running buffer to minimize bulk shifts.

The bulk RI contribution of the sample requires that measurement points (report points), intended to measure the amount of bound analyte, are set before and immediately after an injection, where running buffer flows over the surface. The relative response then reflects the amount bound independent of any bulk contribution (Figure 6).

Figure 6. Contribution of bulk refractive index to the response.

Bulkcontribution

RU

Time

Bulkcontribution

Binding

Dissociation

Amountbound

Baseline



One useful feature of Biacore X100 Control Software is that the bulk contribution can be automatically subtracted. Flow cell 1 can be used as a reference cell for in-line reference subtraction, providing direct recording and display of blank-corrected sensorgrams. The reference curve subtraction automatically corrects for the small time delay between the serial flow cells. A schematic illustration of the principle of reference subtraction is shown in Figure 7.

Figure 7. The effect of reference subtraction. The sensorgram from the reference surface shows the contribution of the bulk, whereas the sensorgram from the active surface shows the actual binding response plus the bulk contribution. In the reference-subtracted sensorgram, only the binding response is shown.

Although the reference subtracted sensorgram shows the actual binding response, it is important to inspect the sensorgrams from both the reference and active surfaces. The sensorgram from the reference surface will, for example, reveal non-specific binding. Remember that non-specific binding cannot be accurately corrected by subtraction of the response on a reference surface, since the binding is not necessarily identical on the active and reference surface. In Exercises 4 and 5, automatic reference subtraction will be used.

RegenerationRegeneration is the process of removing bound analyte from the ligand on the sensor chip surface after analysis of a sample. If the dissociation of the analyte is sufficiently rapid and analyte is completely removed from the surface, no regeneration step is necessary.

Efficient regeneration (removing bound analyte without affecting ligand activity) is crucial to a successful assay. If the regeneration is incomplete or the binding activity of the surface is reduced, the performance of the assay will be impaired and the useful lifetime of the sensor chip will be shortened. Time spent on establishing suitable regeneration conditions is, therefore, a valuable investment.

The choice of conditions for regeneration is dictated by the stability and nature of the ligand and analyte. Our experience has shown that while different applications may need individually tailored regeneration conditions, many surfaces can be regenerated using brief exposure to acidic (glycine-HCl) or basic (NaOH) solutions.


Background informationRegeneration

If the sensor surface is not efficiently regenerated with high or low pH (either because the analyte is not fully removed or because the ligand loses activity), other conditions may be tested alone or in combination with high or low pH. These include:

• Up to 100% ethylene glycol

• High ionic strength (e.g. 5 M NaCl or 4 M MgCl2)

• Low concentrations of SDS ( 0.5%)

Testing regeneration conditionsWhen exploring appropriate regeneration conditions, it is important to test an interval of different conditions, such as pH or ion concentrations, for example. Our recommendation is to use mild conditions first and then increase the harshness of the treatment progressively until suitable conditions are established. In this way, the risk of damaging the ligand on the sensor surface is minimized.

Exploratory testing for regeneration conditions involves injection of analyte over the surface, followed by regeneration solution. The extent to which the analyte is removed is indicated by the baseline response after the injection of regeneration solution. A second injection of analyte is necessary to test whether the ligand is still active and binds the same amount of analyte as before the exposure to the regeneration solution (Figure 8).

Figure 8. Efficient regeneration removes all bound analyte. A second injection of analyte reveals whether the ligand is still fully active.



Injection of regeneration solution often gives a considerable bulk response because the refractive index of the regeneration solution is not matched with the running buffer. The relative bulk response may be either positive or negative depending on the solution used.

The analyte injections described above may be used when you want to establish whether a particular regeneration strategy is worth pursuing. To establish regeneration conditions reliably and reveal trends in regeneration efficiency and ligand activity, it is necessary to perform a series of repeated cycles of analyte injection and regeneration. At least five cycles of analyte binding and regeneration are recommended in testing new regeneration conditions. This regeneration scouting procedure is supported in the Regeneration Scouting wizard in the Biacore X100 Control Software. Once the conditions have been found, more extensive tests may be performed to establish that the assay is stable through large numbers of repeated cycles.

General guidance on how to interpret the trends in analyte binding response and baseline response is given below:

• Ideal regeneration: The analyte response is constant after repeated injections and within ±10% of the level reached in the first injection.

• Too mild conditions: The analyte response decreases and the baseline response increases. The decrease in analyte response results from incomplete removal of the analyte from the surface so that the baseline increases. Test one step harsher conditions or change the type of regeneration solution.

• Too harsh conditions: The analyte response decreases and the baseline response is constant or decreases. The drop in analyte response without a corresponding increase in baseline response indicates that the surface is losing analyte-binding capacity. Change the type of regeneration solution. You may have to prepare a new surface if the ligand has been too extensively damaged.


Background informationRegeneration

Choice of regeneration conditions

Example

This example shows the regeneration scouting for the analyte and ligand included in the Getting Started Kit performed with a Biacore X100 system. To establish appropriate regeneration conditions for the monoclonal anti-2-microglobulin (the ligand) and the 2-microglobulin analyte, injections of analyte followed by regeneration solution were performed repeatedly. Four different glycine-HCl regeneration solutions (pH 3.0, 2.5, 2.0 and 1.5) were examined using five repeated injections.

The results can be seen in Figure 9. At pH 3.0, the sample response(open triangles) decreased and the baseline level (blue diamonds) increased compared to the starting values, indicating that this regeneration solution was too mild. At pH 2.5 and 2.0, the sample responses were stable, returning back to the approximate level of the starting value. At pH 1.5, there was a slight loss of analyte response, indicating that this regeneration solution was too harsh.

This example illustrates that clear-cut differences in the response to different regeneration solutions cannot always be seen. In this case, pH 2.5 was chosen for further testing, although pH 2.0 seemed to give an equivalent effect.

Figure 9. Glycine-HCl solutions with different pH were tested to establish appropriate regeneration conditions for the ligand anti-2-microglobulin and the analyte 2-microglobulin. The baseline response is indicated on the right-hand axis and the sample response on the left-hand axis.


Instrument start up

Instrument start up

This section deals with starting up the system, setting up the liquids required for the run, docking the sensor chip and flushing the liquid system. The priming procedure flushes the liquid system with running buffer. This should be done when a new sensor chip is inserted and/or the running buffer is changed.

Start the system1 Switch on the printer and the PC.

2 Take out the Getting Started Kit from the refrigerator and equilibrate to room temperature.

3 Switch on the instrument at the rear.

The lamps on the front panel light during initiation. When the initiation is ready, Power is lit, the Temperature lamp is lit or flashes, Sensor chip is not lit or flashes and Run is not lit .

power

temperature

sensor chip

run

Lights when power is on. (Green)

Lights when the temperature at the flow cells is stable at the preset temperature. Flashes when the temperature is not sta-ble. (Yellow)

Lights when a sensor chip is docked and ready. Flashes when a chip is inserted but not docked. (Green)

Lights when a run is ongoing. (Green)


Instrument start upBuffer preparation and loading

4 Start the Biacore X100 Control Software from the Windows start menu.

5 In the login-dialog, enter your user name and password and click OK.

6 Ensure that there is fresh buffer or water in the bottle on the left-hand tray and that both buffer tubes are inserted into the bottle.

7 Ensure that the waste bottle on the right-hand tray is empty and the waste tube is inserted in the bottle.

8 The system event log is displayed in the software and you are recommended to perform startup. Click Run now and follow the instructions on the screen.

Buffer preparation and loadingSelect buffer type depending on your specific experiments. For the Getting Started Tutorial you will use HBS-EP+ buffer.

To prepare buffer, dilute 10× buffer solution with degassed deionized water (filtered 0.22 µm). Use degassed water to prevent problems with air bubbles during the run. If the instrument is equipped with the degasser provided with the Biacore X100 Plus Package, you do not need to degas the water.

Always use buffer, fresh for the day and filtered through a 0.22 µm filter to remove particles. A volume of 200 ml is suitable for use during 24 hours.

Prepare buffer in a bottle and place it on the left-hand tray and insert the two buffer tubes. The flow system is filled with buffer automatically when a chip is docked.

If you need to change buffer when the instrument is in standby, stop the standby (Tools:Stop Standby) and change the buffer bottle. When both buffer


Instrument start up

tubes are inserted in the new buffer, select Tools:Prime to fill the system with the new buffer.

Inserting, removing or changing the sensor chip1 Open the upper front door of the instrument.

2 Click the undock chip icon, or select Undock Chip from the Tools menu. When undocking is completed, the Dock Chip dialog is displayed and the sensor chip lamp on the front flashes.

3 Pull out the chip slide (A).

4 If required, remove the old chip (B).

5 Insert the chip with the text on the upper side (C).

6 Push the chip slide all the way in (D).

7 Close the front door and check that the buffer tubes are placed in a bottle of buffer.

8 Fill in the information in the Dock Chip dialog and click Dock Chip.

The chip is docked and the standby flow of buffer over the chip starts automatically.


Instrument start upPreparing and loading samples and reagents

Preparing and loading samples and reagentsDifferent reagents are used depending on the type of run to be performed. The requirements for your assay are displayed in the rack position list in the Biacore X100 Control Software. The list can be printed. If dilution of samples is needed, dilute in degassed running buffer.

The volumes specified in the rack position list are minimum volumes with due consideration for dead volumes.

1 Prepare the samples and reagents in 1.5 ml conical vials (GE Healthcare) according to the volumes and concentrations in the rack position list and cap them to avoid evaporation during the run. Make sure there are no visible air bubbles in the tubes. Use only caps supplied for this purpose by GE Healthcare.

2 Fill the 4 ml vial with water. Always use fresh water filtered through a 0.22 µm filter to remove particles. Do not cap the 4 ml vial! The water is used for needle cleaning during the run and during standby.

3 Click the Load Samples icon.

4 Wait until the lamp Rack locked is switched off.

5 Lift out the rack and place the sample and reagent vials and the water vial according to the rack position list.

Note: Load the rack outside the instrument.

6 Insert the rack in the sample compartment and make sure it sits properly on the rack base.

7 Click OK in the Load Samples dialog.


Biacore X100 Binding Assay


Description of the experimental set upThe following protocol describes the different steps in setting up and evaluating a binding assay in Biacore X100 for the interaction between monoclonal anti-2-microglobulin and 2-microglobulin from the Getting Started Kit.

Experimental materials• Molecular weight of ligand (anti-2-microglobulin): 150 kDa

• Molecular weight of analyte (2-microglobulin): 11.8 kDa

• Concentration in stock solution of ligand: 1 mg/ml

• Concentration in stock solution of analyte: 100 µg/ml (8.5 µM)

Experimental steps• Set up a workflow for binding analysis

• Immobilize monoclonal anti-2-microglobulin via amine coupling

• Run a binding assay for analysis of the interaction between monoclonal anti-2-microglobulin and 2-microglobulin

• Evaluate the results

Note: Step-by step instructions for the binding analysis experiment are also provided in the Support Navigator in the BiacoreX100 Software. To use the tutorial in the Support Navigator click on Learning to use Biacore X100 in the Support Navigator window and then select Getting Started Tutorial to enter the Getting Started section of the Support Navigator.


Biacore X100 Binding AssayDescription of the experimental set up

Requirements• Biacore X100 instrument





ReferencesFor further details refer to:

• Biacore X100 Instrument Handbook

Always read the user documentation before using the system!



Binding Assay set up

Instrument preparations1 If the instrument is switched off, switch it on. Otherwise check in the status

bar at the bottom of the screen that the detection temperature is set to 25°C. Wait until the temperature has stabilized before starting to work with the tutorial.

2 Prepare the reagents in the Amine Coupling Kit according to the instructions provided in the kit. These reagents may be prepared in advance and frozen.

3 Prepare 1X HBS-EP+ running buffer from the 10X stock solution in the Getting Started Kit.

4 If the instrument is in standby mode, choose Stop Standby from the Tools menu.

5 Place the running buffer in a buffer bottle on the tray at the left of the instrument and fit the bottle cap with buffer supply tubes. Make sure both the buffer supply tubes reach the bottom of the bottle.

6 Check that the waste bottle is empty and in place on the tray at the right.

7 Dock an unused Sensor Chip CM5. Note that if you have done the Kinetics Assay exercise before this exercice you can not reuse the chip since different immobilization levels are used in the experiments.

Binding Assay workflow and immobilization set upRunning buffer: HBS-EP+.

Immobilization buffer: Sodium acetate, pH 5.0.

Preparations

The following solutions and vials are required for the immobilization in the Getting Started Binding Assay. Place all solutions except water in 1.5 ml vials and cap the vials.

• EDC and NHS: Prepare as described in the Instructions for Use for the Amine Coupling Kit.

• Ethanolamine: Supplied ready for use in the Amine Coupling Kit.

• Anti-2-microglobulin: Mix 6 µl of ligand stock solution with 194 µl 10 mM sodium acetate pH 5.0.

• Empty: An empty capped vial, used for mixing EDC and NHS immediately before injection.

• Water: Deionized water, placed in an uncapped 4 ml vial.


Biacore X100 Binding AssayBinding Assay set up

1 Start the Biacore X100 Control Software and click the Binding Analysis button at the top of the Control Software screen to start the Create Assay Workflow dialog.

2 Enter anti-b2-micro as your ligand name.

3 Choose My ligand is… : …an antibody and then My antibody is… : …a mouse antibody.

4 The recommended attachment approach is capture using Mouse Antibody Capture Kit. However, for this tutorial you will immobilize the antibody using amine coupling. Check the option Immobilize ligand covalently using Sensor Chip CM5.

5 Click Continue to create and use the workflow.

6 In your folder, click the New Folder icon to create a new sub-folder. Name this folder Tutorial.



7 Enter a name, “Binding Assay”, for the workflow and click Save.

8 A new window opens named Assay Workflow: Binding Assay. The workflow helps you to optimize the experimental steps and provides a framework for your assay.



9 Under the Sensor Surface Preparation part in the Find Immobilization pH box (light blue), click on Enter known values… and enter Sodium Acetate , pH 5.0. (In this exercise the immobilization conditions are known, but to do exploratory pH-scouting in the future you choose Run to find out... .) The entered values will be carried forward as defaults to the immobilization step. You may enter an explanatory comment if you wish. The comment will be shown as a tooltip for the settings overview in the workflow display. Click Save.

10 In the Immobilize box (yellow) click Run and in the Immobilization-Setup dialog check Prime before run. For Flow cell 2, change Aim for immobilized level to Specify contact time. Use the default contact time of 420 seconds. Leave all other settings in the dialog unchanged. Click Next .



11 In the Immobilization - Rack Positions dialog, click Load Samples and wait until the Rack locked indicator on the instrument is turned off. Remove the rack and place the samples in the positions shown in the dialog box. Use the Print Rack Positions option under the Menu button to print a copy of the sample positioning information if you want a hard-copy guide to help you in preparing the rack.

12 Insert the rack in the rack compartment. Click OK in the Load Samples dialog and click Next .

13 In the Prepare Run Protocol dialog, click on Start and then enter a file name for the results, “Binding Immobilization”, and click Save. Note that a link to your immobilization run will appear in the workflow dialog.



Binding Analysis set-upThe unmodified surface in Flow cell 1 will be used as reference (upstream of the active Flow cell 2).

Running buffer: HBS-EP+.

Regeneration solution: Glycine-HCl, pH 2.5.

Preparations

Prepare the sample b2-micro high (85 nM) by mixing 10 µl of the 2-micro-globulin analyte stock solution from the Getting Started Kit with 990 µl running buffer in a 1.5 ml sample vial. Cap the vial. Prepare the sample b2-micro low (8.5 nM) by mixing 100 µl of b2-micro high with 900 µl running buffer in a second 1.5 ml vial. Cap the vial.

Note: Use the same diluted running buffer you prepared earlier when you prepare the samples, not the 10X stock solution in the kit .

1 Continue your workflow with the Assay section.



2 Under the Assay section, in the Find Sample Conditions (light blue) box, click Enter known values… and enter b2-micro low for Name. (In this exercise the sample and regeneration conditions are known, but to run exploratory experiments in the future you choose Run to find out... .)

3 For Contact time, enter 180 seconds.

4 Enter 0 for Dissociation time.

5 Enter 8.5 for Max concentration, and change the concentration unit to nM.

6 Check Regeneration is needed and enter an explanatory comment if you wish. Click Save.

7 In the Find Regeneration Conditions (light blue) box, click Enter known values…



8 In the Regeneration Scouting dialog choose Number of regenerations: 1.

9 Enter Solution: Glycine-HCl pH 2.5.

10 Enter Contact time: 30.

11 Enter Stabilization period: 5 and enter an explanatory comment if you wish. Click Save.

12 In the Run Binding Analysis Assay box (yellow) click Run .

13 In the Binding Analysis – Injection Sequence dialog the settings for Detection and Chip are already chosen for you on the basis of your workflow definition. You will use one sample injection and one regeneration injection, with no enhancement reagent. Click Next .



14 In the Binding Analysis – System Preparation dialog the instrument preparation operations that will be run at the beginning of your experiment are determined. Click Next to accept the default settings and go to the next step. (The conditioning option is not avaliable when you use Sensor Chip CM5.)

15 In the Binding Analysis – Injection Parameters dialog the detailed settings for each injection in the cycle are determined. The values you have entered in the development steps of your workflow will appear as defaults. Click Next to continue to the next step.



16 In the Binding Analysis – Samples dialog the samples that will be run in the assay are set up. Each row in the table represents one analysis cycle.

17 The sample name b2-micro low with concentration 8.5 nM is entered on the first row (taken from the Find Sample Conditions setting). Complete the table for 4 analysis cycles as illustrated below. Each time you enter data in a row, a new empty row is created: however, empty rows do not create analysis cycles. Click Next when you have completed the table.

18 In the Binding Analysis - Rack positions dialog, click Load Samples and wait until the Rack locked indicator on the instrument is turned off.

19 Remove the rack and place the samples in the positions shown in the dialog box. Use the Print Rack Positions option under the Menu button to print a copy of the sample positioning information if you want a hard-copy guide to help you in preparing the rack.

20 Insert the rack in the rack compartment. Click OK in the Load Samples dialog and click Next .

21 In the Binding Analysis - Prepare Run Protocol dialog, click on Start and then enter a file name for the results: “Binding Analysis” and click Save.



Evaluation of Binding dataThe Binding Analysis assay results are opened automatically in the Biacore X100 Evaluation Software. If you want to open the file at a later stage, choose File: Open and select your result file. There are also possibilities to open result files for evaluation from the Biacore X100 Control Software, by selecting the file and click Evaluate, or directly from within the workflow. The steps below will take you through evaluation of the results and illustrate some of the software features.

Examine the sensorgrams1 Choose to view only sample cycles with the Cycle Purpose selector bar.

Click in the sensorgram window to apply the cycle selection.

2 Reference-subtracted sensorgrams are displayed by default. Choose from the Curve Name selector bar to examine the unsubtracted curves.


Biacore X100 Binding AssayEvaluation of Binding data

3 Zoom the display by dragging with the left mouse button. Double-click to return to the previous zoom level.

4 Choose Color by…Sample from the Tools button at the top right of the window, to make it easier to see which sensorgram comes from which sample.

5 Choose Report point: ID and Marker from the Tools button to mark and label report points on the sensorgrams.

6 Choose Sensorgram Adjustment from the Tools button to adjust the sensorgrams for easier comparison. (In this example all curves are adjusted to a common baseline.)



Examine the predefined plotsA set of predefined plots is created automatically when data is opened. Use these plots for quality control and troubleshooting. The plots are accessed in the Plot section of the Evaluation Explorer, at the left of the main screen. Examine each induividual plot as described below.

Baseline. This plot shows the absolute response at the baseline report point, just before the start of the sample injection. Adjust the scale on the baseline plot for a better view. The baseline for the sample cycles should be stable to within about 20-30RU. (Note that the first startup cycle normally differs a lot from the following cycles. This is normal and reflects an adjustment of the Sensor Chip surface to the running buffer.)

Binding level: Sample. This plot shows the relative response just before the end of the sample injection. This is the binding level achieved during sample injection.



Binding stability: Sample. This plot shows the relative response just after the end of the sample injection. Dissociation of the 2-microglobulin used in the Getting Started Tutorial is relatively slow, so that Binding stability values are close to Binding level. In other systems, comparison of these two report points can rapidly identify stable or unstable binders.

Binding to reference: Sample. This plot shows the relative response on the reference surface (flow cell 1) just before the end of the sample injection. These values should not exceed 5-10 RU. Significant responses in this plot indicate that the sample binds to the reference surface, which complicates interpretation of responses observed on the active surface. (Note that in this example, due to the scale, there is an apparent increase in response on the reference surface. This increase is less than 1 RU and is therefore not significant.)



Examine the results as a Bar ChartClick Bar Chart in the toolbar to create a bar chart evaluation item. Binding analysis results are displayed as bar charts of report point values. The default setting shows reference subtracted values for the stability report point for all cycles.

1 Cycles 1-3 are startup cycles and are not relevant for the evaluation. Select cycles 4-7 by dragging with the mouse in the cycles table at the bottom center below the chart.

2 Choose which report point you want to display in the report point list at the bottom right. You can show multiple report points by dragging with the mouse. The Response setting determines whether absolute or relative responses are plotted. Note that the relative response for the baseline report point is zero and will not show in the chart.



3 Click Tools and select Group by…Sample name to sort the chart according to sample name. This makes comparison of replicate measurements very easy.

4 Try the other functions under the Tools button. (Not all will be relevant to these results, but you will get an idea of the settings that are available.)

5 Close the bar chart window. Note that your Bar Chart item has appeared in the Evaluation Explorer. You can reopen it at any time.

End your evaluation1 Choose Save from the File menu to save your evaluation.

2 If you have worked with the Getting Started Tutorial in the Support Navigator, click Leave tutorial at the top of the Support Navigator window.


Biacore X100 Single Cycle Kinetics Assay


Description of the experimental set upThe following protocols describe the different steps in setting up and evaluating a Single Cycle Kinetics assay in Biacore X100 for characterizing the interaction between anti-2-microglobulin and 2-microglobulin from the Getting Started Kit. The described experimental setup requires software version 2.0 or later.

Experimental materials• Molecular weight of ligand (anti-2-microglobulin): 150 kDa

• Molecular weight of analyte (2-microglobulin): 11.8 kDa

• Concentration in stock solution of ligand: 1 mg/ml

• Concentration in stock solution of analyte: 100 µg/ml (8.5 µM)

Experimetal steps• Set up a workflow for kinetic analysis

• Immobilize monoclonal anti-2-microglobulin via amine coupling

• Set up a kinetic assay for characterizing the interaction between monoclonal anti-2-microglobulin and 2-microglobulin using Single Cycle Kinetics approach

• Evaluate the results

Requirements• Biacore X100 instrument





ReferencesFor further details refer to:

• Biacore X100 Instrument Handbook

Always read the user documentation before using the system!


Biacore X100 Single Cycle Kinetics AssayKinetics Assay set up

Kinetics Assay set up

Instrument preparations1 If the instrument is switched off, switch it on. Otherwise check in the status

bar at the bottom of the screen that the detection temperature is set to 25°C. Wait until the temperature has stabilized before starting to work.

2 Prepare the reagents in the Amine Coupling Kit according to the instructions provided with the kit. These reagents may be prepared in advance and frozen.

3 Prepare 1X HBS-EP+ running buffer from the 10X stock solution in the Getting Started Kit.

4 If the instrument is in standby mode, choose Stop Standby from the Tools menu.

5 Place the running buffer in a buffer bottle on the tray at the left of the instrument and fit the bottle cap with buffer supply tubes. Make sure both the buffer supply tubes reach the bottom of the bottle.

6 Check that the waste bottle is empty and in place on the tray at the right.

7 Dock an unused Sensor Chip CM5. Note that if you have done the Binding Assay exercise before this exercice you can not reuse the chip since different immobilization levels are used in the experiments.

Kinetic Assay workflow and immobilization set upRunning buffer: HBS-EP+.

Immobilization buffer: Sodium acetate, pH 5.0.

Preparations

The following solutions and vials are required for the immobilization in the Getting Started Kinetics Assay. Place all solutions except water in 1.5 ml vials and cap the vials.

• EDC and NHS: Prepare as described in the Instructions for Use for the Amine Coupling Kit.

• Ethanolamine: Supplied ready for use in the Amine Coupling Kit.

• Anti-2-microglobulin: Mix 6 µl of ligand stock solution with 194 µl 10 mM sodium acetate pH 5.0.

• Empty: An empty capped vial, used for mixing EDC and NHS immediately before injection.

• Water: Deionized water, placed in an uncapped 4 ml vial.



1 Start the Biacore X100 Control Software and click the Kinetics/affinity… button to enter the Create Assay Workflow- Kinetics/Affinity window.

2 Enter Ligand name: anti-b2-micro , My ligand is… : …an antibody and My antibody is… : …a mouse antibody. You will now see a preview of a recommended Assay Workflow.



3 Under Ligand attachment approach check Immobilize ligand covalently using Sensor Chip CM5. Click Continue and save your Workflow as “Kinetic Assay”.

4 A new window opens named Assay Workflow: Kinetic Assay. The workflow helps you to optimize the experimental steps and provides a framework for your assay.



5 Under the Sensor Surface Preparation part in the Find Immobilization pH box (light blue), click on Enter known values… and enter Sodium Acetate , pH 5.0. These values will be carried forward as defaults to the immobilization step. You may enter an explanatory comment if you wish. The comment will be shown as a tooltip for the settings overview in the workflow display. Click Save.

6 In the Immobilize box (yellow) click Run and in the Immobilization-Setup dialog check Prime before run. Under Flow cell 2, select Method: Amine and check Aim for immobilized level with Target level: 1200 (RU) and Wash solution: Ethanolamine. Click Next .



7 In the Immobilization- Rack positions dialog, click Load Samples and wait until the Rack locked indicator on the instrument is turned off.


9 Insert the rack in rack compartment. Click OK in the Load Samples dialog and click Next .

10 In the Prepare Run Protocol dialog, click on Start and then enter a file name for the results, “Immobilization Exercise”, and click Save.

11 When your immobilization is finished the results and sensorgrams will be shown. Observe your results and click Close in both windows to get back to your Kinetic Assay workflow. Note that the results of your immobilization run will be available as a link in your workflow.



Kinetic Assay set-upThe unmodified surface in Flow cell 1 will be used as reference (upstream of the active Flow cell 2).

Running buffer: HBS-EP+.

Regeneration solution: Glycine-HCl, pH 2.5.

Preparations

Dilute the analyte 2-microglobulin in running buffer to 128 nM (9 µl 2-microglobulin + 591 µl running buffer). Prepare the concentration series from the 128 nM sample: mix 150 µl of the 128 nM solution with 450 µl running buffer to get the 32 nM concentration. Mix 300 µl of the 32 nM concentration with 300 µl running buffer to get the 16 nM concentration. Continue the dilution series to obtain the following concentration series: 32, 16, 8, 4 and 2 nM.

Note: Use the same diluted running buffer you prepared earlier when you prepare the samples, not the 10X stock solution in the kit.

1 Continue your Kinetic Assay workflow with the Assay section.



2 Under the Assay section, in the Find Sample Conditions (light blue) box, click Enter known values… and enter Name: b2-micro, Contact time: 120 (s), Dissociation time: 600 (s) and Max concentration: 32 nM. Check Regeneration is needed and enter an explanatory comment if you wish. Click Save.

3 In the Find Regeneration Conditions (light blue) box, click Enter known values…



4 In the Regeneration Scouting dialog enter Number of regenerations: 1, Solution: Glycine-HCl pH 2.5, Contact time: 30 (s) and Stabilization period: 0 (s). Enter an explanatory comment if you wish and click Save.

5 In the Run Kinetics/Affinity Assay box (yellow) click Run.

6 In the Kinetics/Affinty – Injection Sequence dialog the settings for Detection and Chip are already chosen for you on the basis of your workflow definition. You will use the checked Kinetics type: Single-cycle and one regeneration injection. Click Next .



7 In the Kinetics/Affinty – System Preparation dialog check Prime before run. Also check Run startup cycles and enter Solution: Buffer and Number of cycles: 1. Click Next .

8 In the Kinetics/Affinty – Injection Parameters dialog the detailed settings for each injection are determined. The values you have entered in the development steps are entered by default. Verify the data and click Next .

9 In the Kinetics/Affinty – Samples dialog example values are pre-set with two zero concentration cycles and one analyte cycle. Enter 11800 in the MW (Da) column and 2 in the Dilution column. The dilution factor is set to 5 by default to cover a broader concentration range, but since the working range for 2-microglobulin has already been determined you will use a dilution factor of 2 in this assay. A concentration series will be created automatically. Click Next .



10 In the Kinetics/Affinity- Rack positions dialog, click Load Samples and wait until the Rack locked indicator on the instrument is turned off.


12 Insert the rack in the rack compartment. Click OK in the Load samples dialog and click Next .

13 In the Kinetics/Affinity - Prepare Run Protocol dialog verify that all preparations are completed. Click on Start and then enter a file name for the results: “Kinetics Assay” and click Save.

14 When your kinetic run is finished the results will be shown in the Biacore X100 Control Software and the Biacore X100 Evaluation Software will start automatically with your result file open.


Biacore X100 Single Cycle Kinetics AssayEvaluation of Kinetic data

Evaluation of Kinetic dataYour result file, “Kinetic Assay”, will be automatically opened in the Biacore X100 Evaluation Software. If you want to open the file at a later stage, choose File: Open and select your result file. There are also possibilities to open result files for evaluation from the Biacore X100 Control software, by selecting the file and click Evaluate, or directly from within the workflow. The steps below will take you through evaluation of the results and illustrate some of the software features.

1 In the Evaluation Explorer (left column) you will have a list of Evaluation items. These are pre-defined evaluation items and quality control plots. Additional evaluation items will also be listed as they are created.

2 Click on the Kinetics/Affinity button on the toolbar or choose Evaluation: Add Kinetics/Affinity.



3 The Select Curves dialog presents the cycles available in the current results set. Choose the cycles to be included in the evaluation in the Include column. By default, all reference-subtracted sensorgrams are included. The sensorgrams for the samples are shown in color, and sensorgrams for blanks (zero concentrations) in light gray. Click Next .



4 The Select Data dialog shows the blank subtracted curve set, i.e. the sensorgrams from the zero concentrations have been subtracted to correct for any systematic disturbances. Here it is possible to delete selected regions to eliminate spikes or other disturbances. To remove regions, mark the area to remove by dragging with the right mouse button. Adjust the left and right borders of the selection if necessary and click Remove Selection. If you wish to restore removed data, click Undo. You can restore any number of data removal operations.


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5 Click on Kinetics to initiate the kinetic evaluation. Use the model 1:1 Binding from the pull-down list. Click on Fit to perform the fitting.

6 The Quality Control tab (for evaluations using the predefined 1:1 Binding model only) gives a brief overview of the reliability of the results.



The symbols used on this tab have the following meanings:

Use the Quality Control assessment as a help in making your own judgement of the results. Pass status in the quality control parameters does not necessarily indicate that the fit is acceptable or that the results are biologically relevant. On the other hand, Fail status in any of the parameters is a reliable warning indicator. Base your assessment on the overall quality of the results and the fitting, taking all quality control parameters into account.

Note: The recommended procedure on how to assess the fitted results are described in detail in the Support Navigator and the following steps gives you a brief introduction to this procedure.

7 Click on the Report tab and inspect the report window. The report window displays the parameters ka, kd, KD, RI, Rmax, tc and kt. Always assess if the calculated numbers are biologically and experimentally relevant. More information regarding these parameters and the statistical parameters (Chi2 and U-value (and Standard Error (SE) or T-value listed under the Parameters tab) can be found in the Support Navigator and in the Biacore X100 Handbook.

(Green) Pass: quality assessment acceptable.

(Yellow) Warning: quality assessment close to the limits of acceptability

(Red) Fail: quality assessment unacceptable

(Blue) Neutral or user assessment required


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8 Inspect the sensorgrams. The black curves superimposed on top of the sensorgrams represent the calculated curves.

9 Switch to the residual plot window by clicking on the Residuals tab. The residual plot shows the difference in RU between each data point for the experimental curves and the calculated curves. The shape and distribution of the residuals indicate how well the data fit to the chosen model. As an aid in judging the residuals, guidelines are drawn on the residual plot to indicate the range of acceptability. Most of the residuals should be within the inner (green) limits.

10 Click on Finish to complete the evaluation and place the item in the evaluation explorer panel.



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ContentsBiacore X100 Getting StartedIntroductionRequirementsReferencesContents of the Getting Started KitOrdering information

Background informationBiacore terminologySensor surface propertiesImmobilizationAmine couplingConditions for immobilizationImmobilization pH-scoutingImmobilization levelsLigand requirements

Interaction analysisSample requirements

RegenerationTesting regeneration conditionsChoice of regeneration conditions

Instrument start upStart the systemBuffer preparation and loadingInserting, removing or changing the sensor chipPreparing and loading samples and reagents

Biacore X100 Binding AssayDescription of the experimental set upExperimental materialsExperimental stepsRequirementsReferences

Binding Assay set upInstrument preparationsBinding Assay workflow and immobilization set upBinding Analysis set-up

Evaluation of Binding dataExamine the sensorgramsExamine the predefined plotsExamine the results as a Bar ChartEnd your evaluation

Biacore X100 Single Cycle Kinetics AssayDescription of the experimental set upExperimental materialsExperimetal stepsRequirementsReferences

Kinetics Assay set upInstrument preparationsKinetic Assay workflow and immobilization set upKinetic Assay set-up

Evaluation of Kinetic data

biacore x100 gs bookwater vials, 4 ml (package of 1000 vials) br-1006-54. biacore x100 getting...

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